The risk from pathogenic microorganisms in foods has been recognized for many years, and bacterial agents are generally implicated as the contaminants. Food-borne disease may be one of the most notable public health problems. The rapid detection and identification of pathogenic microorganisms in foods, and its manufacturing environment, is of utmost importance if we are to develop and implement control and prevention strategies leading to a safer food supply. From 1993–1997, 2,751 outbreaks of food-borne illness were reported to the CDC. These outbreaks accounted for 86,058 persons affected. Bacterial pathogens caused the largest percentage (75%) of these reported outbreaks. The predominant cause in the reported cases was Salmonella Enteritidis, thought to have originated in egg products. Additionally, multi-state outbreaks of Eschericia coli 0157:H7 contributed significantly to the total figures for morbidity and mortality (1). Listeria monocytogenes, a gram-positive contaminant, is an emerging public health threat to the safety of food products as well.
In addition to issues related to food safety, antibiotic-resistant bacteria, including Gram-positive bacteria, are becoming an increasing issue in U.S. hospitals and communities. Community-acquired pneumonia strikes approximately four million Americans each year and hospitalizes about 600,000. Approximately 500,000 cases of community-acquired pneumonia each year are the result of infection with S. pneumoniae, as shown in the New England Journal of Medicine (1995; 333:1618–1624). Resistance to penicillin, the most common agent used to treat S. pneumoniae, now approaches 40 percent. Additional resistance has been reported against cephalosporins and non-beta-lactam agents, and nearly half of these strains can be classified as highly resistant. High-dose penicillin and cephalosporins remain first-line therapies, however, a broader range of agents is needed. Vancomycin, the next generation of fluoroquinolones with agents such as sparfloxacin, the new streptogramin class, as well as combination therapies, will help physicians stay one step ahead of resistant pneumococci.
The gram-positive pathogens, penicillin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci, complicate the treatment of serious infections and have been linked to extended hospitalizations, higher medical costs and high mortality rates. Drug-resistant Streptococcus pneumoniae poses a growing threat to people in places where they live and work. Streptococcus pneumoniae infections—including pneumonia, sinusitis, meningitis and otitis media—are among the leading causes of death and illness among the elderly, young children and persons with underlying medical conditions. Drug-resistant S. pneumoniae often strikes vulnerable patient populations in daycare settings, nursing homes and prisons.
Staphylococcus aureus, the most common cause of more than a dozen conditions in both hospitals and communities, often colonizes without any sign of infection, and then from this reservoir gains access to skin and deep tissue, where it subverts the immune system. Staphylococcal infections range from local skin infections to endocarditis (heart valve infection), osteomyelitis (bone infection) and sepsis (blood stream infection). Methicillin-resistant S. aureus first emerged in the early 1960s. Several strains of S. aureus are now resistant to a wide variety of currently available antibiotics, including penicillins, macrolides, fluoroquinolones and lincosamides.
In the same bacteria family, multidrug-resistant Staphylococcus epidermidis also compromises patient health, and has been established as a leading cause of hospital-acquired bloodstream infections. More than 80 percent of S. epidermidis isolates in U.S. hospitals are methicillin resistant, and recent studies have found resistance to quinolones, cephalosporins and vancomycin. This drug resistance is a growing concern, particularly for immunocompromised cancer patients.
Vancomycin is considered the agent of last resort for Gram-positive infections. Vancomycin-resistant enterococci, an increasingly frequent cause of hospital-acquired infections in the United States, are resistant to virtually all currently available antibiotics including vancomycin.
Accordingly, there is a significant need in the art for an effective method of detecting and diagnosing these pathogens. Unfortunately, to date, testing bacteria, yeast and fungi has been excessively time consuming and labor intensive. While the onset of symptoms from endotoxin from coliform bacteria may be exceedingly rapid, laboratory based diagnosis will typically take days. The present techniques used to detect the presence of bacteria involve aseptic transfer of a sample, streaking the sample having bacterial organisms on agar plates after serial dilution, and colony enumeration. This laborious and lengthy process requires at least 24 to 48 hours for a positive result and substantially longer for a negative result.
Both the detection and characterization of microbial contaminants in food and water samples have historically relied upon the use of bacterial enumeration techniques, both in liquid and solid culture media. These methods, while sufficiently sensitive to detect a small number of viable organisms, require lengthy sample preparation time. The use of ELISA techniques and nucleic acid hybridization probes, while accurate, have less sensitivity, and therefore require lengthy isolation and enrichment periods to reach the analytical detection limits for these techniques. Therefore, there is a need for a method of determining cell numbers that is fast as well as sensitive.
Other analyte tests require an organism to ingest a detectable material, such as fluorescein. In yet other tests, an antibody, specific for an antigen on the target bacteria is labeled with fluorescein to make a fluorescent antibody. Another approach involves use of a visualization polymer coupled to a detecting agent that binds the target organism, wherein the visualization polymer is made up of detectable visualization units, such as multiple enzymes or labeled polyolefins, which are directly or indirectly bonded together (see, e.g., U.S. Pat. No. 4,687,732 to Ward et al.). Another approach involves covalent conjugation of polymyxin B (PMB) and an enzyme reporter molecule, such as horseradish peroxidase (HRP), to produce a complex for use in a binding assay to detect the target organism (Applemelk et al. (1992) Anal. Biochem. 207:311–316). An organic “chemical tag” that comprises populations of binding agents and detectable labels has also been described (Olstein et al., U.S. Pat. No. 5,750,357).
However, all of the aforementioned labeling methods suffer from the inherent steric interference introduced by the size of the tag, typically larger than 100 D3, primarily contributed by the reporter group, usually an enzyme. By contrast, the antibiotic usually being a substantially smaller molecule (20 D3) than the macro-molecular complexes described above, can readily penetrate membrane-bound receptors on the cell surface. Consequently, a continuing need exists for a sensitive and rapid method to detect extremely small amounts of target biological analytes.
Antibiotics have been used primarily as therapeutic agents and growth promoting substances. However, there is evidence in the literature for their use for diagnostic purposes (2,3). Many methods for conjugation of reporter groups to antibiotic compounds are frequently unsuitable, for both technical reasons, such as loss of biological activity, loss of solubility and economic, i.e. the cost of enzymes, dyes and the conjugation chemistry. Chemiluminescent labeling of macromolecules has been demonstrated to yield greater analytical sensitivity than the use of many fluorescent probes because of simplicity of the optics resulting in lower background signal (4).
Therefore, there is a need in the art for detection methods for pathogenic organisms. Ideal methods would utilize small reporter groups and provide sensitive detection. There is a further need to preferentially detect viable organisms, as non-viable organisms may not of themselves provide a threat to the health of an individual and may not indicate the source of any potential danger, particularly where bacteria are a food contaminant. Further, there is a need to distinguish over any background signal of non-viable pathogens in order to accurately determine the numbers of live cells.